Field of the Invention
The invention relates to a mass spectrometric method of determining microbial resistances to antibiotics using isotopically labeled nutrient components.
Description of the Related Art
Instead of the statutory “unified atomic mass unit” (u), this document uses the unit “dalton” (Da), which was added in the last (eighth) edition of the document “The International System of Units (SI)” of the “Bureau International des Poids et Mesures” in 2006 on an equal footing with the atomic mass unit; as is noted there, this was done primarily in order to be able to use the units kilodalton, millidalton and similar.
For reasons of simplicity, only the term “proteins” is used in this document, although in the mass range considered here it would often be better to call the proteins “peptides”. The transition from the lighter peptides to the heavier proteins is fluid, however, and not precisely defined.
The term “antibiotic” here covers pharmacologically active substances for the treatment of bacterial infectious diseases and other antibacterial substances, for the purpose of disinfection, for example.
The successes of penicillin, but also the appearance of the first resistances, led researchers to search for and discover many more antibiotics. Ever since penicillin was used as the first pharmacologically active substance, bacterial strains have increasingly developed various types of resistance, i.e. they have acquired characteristics which allow them to weaken the effect of antibiotic substances or to neutralize it completely. Resistances are now widespread: in the USA, around 70% of the infectious germs acquired in hospitals are resistant to at least one antibiotic. Patients are often infected with bacterial strains which are resistant to several antibiotics (multi-resistance). The so-called problematic germs are mainly methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas spec., Escherichia coli with ESBL resistance and Mycobacterium tuberculosis. The CDC (Center for Disease Control and Prevention) estimates that two million infections were acquired in hospitals in the USA in 2004, with around 90,000 deaths.
The reasons for the increase in resistances are manifold: irresponsible prescription of antibiotics, even when not necessary; courses of treatment which are irresponsibly broken off; irresponsible, often purely preventative usage in agriculture and animal husbandry. All these types of behavior help to select and spread resistant bacterial strains, as opposed to non-resistant bacterial strains.
The success of a therapy for bacterial infections, which can be life-threatening in acute situations, such as sepsis, or as a secondary infection during an existing primary illness (or primary infection), often depends on the first administration of an antibiotic being effective. Targeted administration requires not only that the pathogen is identified as quickly and correctly as possible, but also that its resistance to different antibiotics is determined quickly.
In routine microbiological work nowadays, the resistance of microbes is determined by culturing microbes from a sample under investigation in vitro on nutrient media (e.g., agar plates) or in nutrient media (e.g., culture broths), to which an antibiotic is added in each case. Whether the microbes multiply under the influence of the antibiotic, i.e., are resistant to the antibiotic, is determined visually by eye or in an automated process using optical devices. The routine method based on an optical evaluation allows a simple determination of the resistance, but it is time-consuming because the cultivation has to be carried out until an optically discernible effect is achieved. This method usually takes 24 to 48 hours. An advantage of this routine method consists in the fact that the efficacy or inefficacy of an antibiotic against the microbes of a sample is measured directly (it is a “functional test”).
The usual procedure is to test cultures with graduated concentrations of the antibiotic in order to determine the “minimum inhibitory concentration” (MIC). The minimum inhibitory concentration designates the lowest concentration of a substance at which the multiplication of a microorganism can no longer be perceived visually. The usual practice is to determine the MIC, but some antibiotics can also be characterized via the “minimum bactericidal concentration” (MBC), where 99.9% of the germs are killed within a fixed period of time. While the MIC can be determined, in principle, for every antibiotic, the MBC only makes sense for those antibiotics which can develop not only an inhibitory, but also a bactericidal effect. These are aminoglycosides, gyrase inhibitors and penicillins, for example. On the basis of the resistance determined, the detected germs are termed S—sensitive, I—intermediate or R—resistant.
In addition to culturing in the presence of antibiotics, there are also genetic methods to determine resistances. Here, a resistance is determined by detecting known resistance genes in the genome of the pathogen in question. An advantage of the genetic methods consists in the fact that the resistance genes can be amplified by techniques such as polymerase chain reaction (PCR), and thus the time needed for the analysis is no longer determined by the growth rate of the bacteria. The disadvantages are that they are more expensive than routine methods and are not functional tests. A resistance gene may be present, but not be expressed, which means the bacterial strain under investigation is not resistant, but the method detects it as being resistant.
Many species of microorganism, particularly bacteria and unicellular fungi such as yeasts, can be identified by mass spectrometry nowadays—quickly and with low error rates. The term “identification” here means taxonomic classification, i.e. the determination of family, genus and species. In routine laboratory work, the identification is achieved by means of a similarity analysis between a MALDI mass spectrum (MALDI=Matrix Assisted Laser Desorption/Ionization) of the sample under investigation and MALDI reference spectra of known microorganisms. In the similarity analysis, each reference spectrum is assigned a classification number, which is a measure of the agreement between the corresponding reference spectrum and the mass spectrum of the sample. If the similarity values exceed certain threshold values, family, genus, species and even strain can be identified. This method for identifying microorganisms has proven to be extraordinarily successful, both in large-scale studies and in the daily routine in many microbiological laboratories. Depending on the instrument, 48 to 384 microbial samples can be determined at the same time. After cultivation of a colony, it takes only minutes to identification. It is thus a fast and low-cost method having very low error rates, far lower than conventional microbiological identification methods. Recent studies confirm that this mass spectrometric identification method is more reliable at providing correct results than DNA analysis, which has been deemed to be the “gold standard” to date. There are meanwhile mass spectrometers, associated evaluation programs, and libraries of reference spectra on the market which are certified as IVD products for medical diagnostics in accordance with the German Medical Devices Act (MPG) and other, national and international regulations and guidelines.
Attempts have been made to extend the mass spectrometric identification of microorganisms to a mass spectrometric determination of their resistances. Unfortunately, determining resistances directly from a mass spectrum has so far only proved possible in rare exceptional cases, even though the resistances should also be detectable from the presence of new or modified proteins.
The patent specification DE 10 2006 021 493 B4 (V. M. Govorun and J. Franzen, 2006, corresponding to GB 2438066 B, U.S. Pat. No. 8,293,496 B2; called “Govorun” in the following) discloses mass spectrometric methods for determining the resistance of bacteria, in which protein profiles of the bacteria are measured by mass spectrometry after cultivation with and without added antibiotics and compared. As the patent specification explains, the resistance can, for example, be determined by the fact that the microbes continue to live and take up nutrient from the medium even in the presence of antibiotics. If the medium contains isotopically labeled nutrient components, the resulting change in the mass spectra indicates the resistance. Non-resistant microbes, on the other hand, suffer a growth inhibition or a structural destruction and no longer take up such nutrient components. Like the routine method described above, Govorun's method is a functional test, but is faster at providing measurable information than the standard optical methods.
An embodiment of the Govorun method is described in the article “Establishing Drug Resistance in Microorganisms by Mass Spectrometry”, (P. A. Demirev et al.; J. Am. Soc. Mass Spectrom. (2013)). Microbes are cultivated, with the addition of antibiotics, in a medium in which all 12C atoms have been replaced by 13C. The possible shifts of mass spectrometric peaks (mass signals), which might occur in the mass spectra of the microbes from these cultures compared to the usual reference spectra, are calculated in advance, at least approximately, by means of four different methods. In the simplest method, an approximate advance calculation uses the average content of C atoms for proteins of a given mass; more complicated methods are directed to a de-novo sequencing by tandem MS or to an identification with the aid of protein databases. These shifted peaks should only be found where there is a resistance, because only resistant microbes can grow when antibiotics are present. This method is decidedly expensive, however, because it uses a completely isotope-labeled medium; in addition, the prediction of the peak shifts is either imprecise or elaborate.
In the patent application WO 2011/152899 A1 (P. A. Demirev et al.), as stated in the abstract, mass spectra of microbes, or of isolated biomarkers from microbes which have grown in an isotope-labeled medium with an antibiotic, are compared with mass spectra from microbes or biomarkers from microbes which have grown in normal media without antibiotic. The resistance is determined by predicting and detecting a characteristic mass shift, which indicates that the microbe grows in the presence of an antibiotic and takes up isotopically labeled material in one or more biomarkers, causing the mass shift.
There is an ongoing effort to provide a mass spectrometric method with which the resistance of microbes to one or more antibiotics can be determined relatively quickly (preferably in less than eight hours), with certainty and, most importantly, at low cost also. It should preferably be possible to carry out the method in a routine mass spectrometer, which is also used for identifications.